Oral Cholera vaccine campaign in Sudan

A pre-emptive mass immunization campaign with oral cholera vaccines [OCV] has recently been carried out in Sudan in order to prevent spread of cholera amongst refugees escaping from South Sudan on account of war. The campaign, completed in two rounds, ended on 1st of September 2016

Ensayo clínico de reto, para evaluar una cepa candidata a vacuna contra el cólera / Challenge clinical trial for evaluation of a vaccine candidate strain against cholera

INTRODUCCIÓN: la cepa atenuada 638 Vibrio cholerae O1 El Tor, Ogawa, ha demostrado ser bien tolerada e inmunogénica por vía oral en estudios realizados en voluntarios sanos. OBJETIVO: se evaluó la protección conferida contra el cólera, en un ensayo clínico de reto, para el escalado tecnológico y farmacéutico de este candidato vacunal como ingrediente activo a escala industrial. MÉTODOS: en el estudio participaron 21 voluntarios sanos, 12 de ellos recibieron el candidato vacunal, y 9 ingirieron un placebo; 28 d después, todos recibieron una dosis infectante de una cepa virulenta de V.cholerae. RESULTADOS: la diarrea se registró en 7 de los 9 placebos, mientras que ninguno de los voluntarios vacunados presentó diarrea. Los voluntarios placebos del grupo sanguíneo O, tuvieron diarreas con mayor frecuencia e intensidad. Todos los voluntarios en el grupo placebo excretaron V. cholerae mientras que solo 3 (25 por ciento) de los 12 vacunados la excretaron. CONCLUSIONES: en este modelo de ensayo de reto, la cepa 638 demostró proteger contra la diarrea producida por una cepa virulenta de V. cholerae.

INTRODUCTION: live attenuated oral Vibrio cholerae O1 El Tor, Ogawa strain 638 has demonstrated to be well tolerated and immunogenic when administrated orally in studies carried out in healthy volunteers. OBJECTIVE: to evaluate the protection against cholera infection in a challenge clinical trial, for the technological and pharmaceutical scale-up of this vaccinal candidate as active ingredient at industrial level. METHOD: a total of 21 healthy volunteers were involved in this trial; the vaccine candidate was administered to 12 of them and the remaining nine were given the placebo. Twenty eight days later, all of them received an infective dose of a V. cholerae virulent strain. RESULTS: diarrheas were observed in 7 out of 9 placebos whereas not a single vaccinated volunteer showed diarrheas. More frequent and intense loose stools were found in the placebo volunteers with O-blood group. All volunteers in he placebo group excreted V. cholerae, but only three (25 percent) out of the 12 vaccinated volunteers did so. CONCLUSION: in this challenge clinical trial model, the 638 strain proved to protect people against the diarrhea caused by a virulent V. cholerae strain.

Comparative stuy for LPS Extraction of NAG vibrio cholerae by EDTA, heating method and hot phenol method

Vibrio cholerae [NAG] was isolated from patients with diarrhea, identification by microbiological, biochemical and serological techniques, then Lip polysaccharide was extract from Vibrio cholerae [NAG][31G, 11G, 24G, 13G] by phenol and EDTA-heating method for comparison, The extracts were purified by Sepharose-4B gel. The results showed that EDTA-heating method was efficient and less expensive of time and material.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Rapid diagnosis of cholera by coagglutination test.

In this study the coagglutination test for the rapid diagnosis of cholera is evaluated in comparison with the conventional culture method. A total of 553 stool specimens were processed from cases of acute gastro-enteritis. The sensitivity and specificity of coagglutination test was 92.77% and 95.65% respectively. The coagglutination test is found to be simple, reliable and rapid method for the diagnosis of cholera.

Desarrollo de vacunas basadas en la atenuación racional de bacterias: la experiencia con salmonella typhi y vibro cholerae

Una de las observaciones pilares en el control de enfermedades infecciosas, es el hecho de que muchas cepas microbianas atenuadas, es decir, debilitadas en su capacidad de producir enfermedad, conservan su habilidad de inducir una respuesta inmune protectora. Esto las convierte en excelentes candidatos para ser utilizadas como vacunas. El conocimiento más profundo de los mecanismos de virulencia y patogenicidad microbianos, sumado al rápido desarrollo de técnicas de manipulación genética de las últimas decádas nos han provisto con las herramientas necesarias para llevar acabo una atenuación más precisa de microorganismos. Al mismo tiempo han garantizado un acercamiento más cauteloso al desarrollo de vacunas, en el que el riesgo de reversión al fenotipo silvestre, ha sido dramáticamente minimizado.Este artículo quiere dar, a través de dos ejemplos, una idea de como la atenuación racional puede conducir al desarrollo de vacunas más efectivas y seguras contra enfermedades infecciosas de alta incidencia en Latinoamerica, tales como cólera y fiebre tifoidea

Análise dos anticorpos vibriocidas e aglutinantes na população urbana do Município do Manacapura/AM (1992/1993) / Vibriocidal and agglutinating antibodies in the urban population in the municipality of Manacapuru/AM (1992-1993)

Foi realizado um estudo sorológico em 1.196 indivíduos residentes na área urbana do município de Manacapuru/AM, visando analisar o perfil dos anticorpos vibriocidas e aglutinantes. Empregou-se o procedimento de amostragem aleatória sistemática na obtenção da amostra populacional. Um ano após, obteve-se uma 2ª amostra de soro de 120 indivíduos (10% da amostra inicial), escolhidos aleatoriamente entre os participantes do inquérito, com o objetivo de avaliar o comportamento dos anticorpos nesse intervalo de tempo. Foram empregados os métodos de microtitulação de anticorpos vibriocidas e de soroaglutinação em tubos. A associação entre os anticorpos estudados foi determinada pelo coeficiente de correlação, calculado com base na distribuição de freqüência dos títulos detectados. A análise dos resultados revelou forte correlação positiva entre os anticorpos (r = 1,0) e queda nos títulos em grande proporção das amostras após um ano.

A serological study was carried out involving 1,196 individuals residents in the urban area of Manacapuru--Amazonas, to evaluate the behavior of vibriocidal and agglutinating antibodies. A systematic random sampling procedure was employed to obtain the sample. A year later a 2nd sample of serum was obtained from 120 individuals selected among the participants of the survey. Vibriocidal antibodies microtitulation and seroagglutination in tubes were employed. The correspondence between the studied antibodies was determined by the correlation coefficient calculated according to the frequency of the titles detected. The analysis of the results revealed positive correlation between the antibodies (r = 1.0) and a decrease in titles in a large proportion of the positive samples one year later.

Rapid identification of vibrio cholera-01 by coagglutination test using mono-specific antibody

Mono-specific antisera against Vibrio cholera Ogawa NIH-43 and Vibrio cholera Inaba 35-A3 were prepared from rabbit hyperimmune sera by absorbing against a heterologous strain. Using ammonium sulphate precipitation procedure, gamma globulins were purified and concentrated. To visualize antigen-antibody reaction, gamma globulins were conjugated to Staphylococcus aureus cowan-1 [NCTC: 8325] in the presence of 50% propanol-1. Then equal suspensions of each conjugated serum were mixed to prepare V. cholera. Rectal swab samples from suspected choleric patients were inoculated in bile peptone broth for 5 hours at 37°C. One drop of each sample was mixed with one drop of VBCR and coagglutination was read at 2-3 minutes. The results were compared with corresponding results obtained from conventional culture methods. Specificity and sensitivity of coagglutination tests were found to be 98.03% and 95.1%, respectively. Regarding the fact that rapid diagnosis of cholera is vital to save patients, our study reveals that coagglutination test, using bivalent mono-specific antisera, can be considered as a simple, rapid and reliable test to detect V. cholera-01 from stool samples of suspected patients

Relaciones fenotípicas y moleculares entre cepas de vibrio cholerae 01 aisladas en Chile, Perú y Bolivia: comparación con cepas de reservorios ambientales / Phenotypical and molecular relation between vibrio cholerae 01 strains, isolated in Chile, Perú and Bolivia: comparison with strains from environmental reservoirs

The phenotype, biotype and susceptibility to nine antimicrobials was determined for each isolated strain. Also, the genes of cholera and termolabile toxins were determined using DNA probes and a chromosomal restriction profile was done using HindIII, EcoRI and NotI enzymes. Features studied were similar in the 53 strains isolated from patients. Those isolated from environmental reservoirs had different antimicrobial susceptibility, showing ampicillin resistance and the GT gene was detected in one of 20 strains, compared to clinical samples were it was present in all. Strains isolated from patients and envirinment had similar chromosomal restriction profiles. The chromosomal restriction profile gives an image of bacterial genome and it is a useful and reliable tool for the epidemiological surveillance of cholera

CAMP test for the identification of Vibrio cholerae 0139.

The confirmation of the identity of Vibrio cholerae serogroup 01 and serogroup 0139 is usually done by slide agglutination tests using specific antisera. Antiserum to V. cholerae 01 is freely available but not antiserum to V. cholerae 0139, thus making specific identification of the latter more difficult. A modified CAMP (Christie Atkins and Muench - Paterson) test has been described as a possibility in the identification of V. cholerae 0139 and we have evaluated this on 197 strains of organisms including 48 V. cholerae 0139, 50 V. cholerae 01, 49 non-01, non-139 V. cholerae and 50 Aeromonas species. The test had a sensitivity of 98.9 per cent and a specificity of 92.9 per cent in the identification of these strains, when compared with faeces culture as the gold standard.

Seguridad e inmunogenicidad de la vacuna oral contra el cólera: estudio en 20 voluntarios / Safety and immunogenicity of the oral anti-cholera vaccine againts the cholera: Study in 20 volunteers

Se realizó un estudio de seguridad e inmunogenicidad de la vacuna de células enteras de Vibrio cholerae 01 más la subunidad B recombinante de la toxina (CE/sBr) en 20 voluntarios sanos, en el Laboratorio de Microbiología del Instituto Nacional de Salud. Se suministraron tres dosis de la vacuna con intervalos de dos semanas entre cada una y se recolectaron muestras de sangre el día de la primera dosis y semanalmente durante ocho semanas. Se anotaron los efectos secundarios durante los cinco días siguientes a la ingestión de cada una de las dosis. En los sueros, se determinaron los niveles de anticuerpos antibacterianos empleando la técnica vibriocida y los de anticuerpos antitoxina de las clases IgG e IgA empleando la técnica de ELISA. Se presentaron efectos secundarios leves como náuseas, cefalea y malestar abdominal después de cada una de las dosis en 4, 5 y 2 de los voluntarios. El porcentaje de seroconversión más alto para títulos vibriocida fue del 50 por ciento y la mayor media geométrica fue de 21,38 (15,3-29,85) ambos eventos en la tercera semana. El porcentaje máximo de seroconversión para los anticuerpos antitoxina fue del 84 por ciento en la octava semana para IgG y del 89 por ciento en la sexta semana para IgA y la media geométrica fue de 245,5 (198-305) en la sexta semana para IgG y de 74,13 (60-98) en la tercera semana para IgA. Con estos datos podemos concluir que la vacuna CE/sBr fue segura e inmunogénica en los voluntarios estudiados

Rapid detection of Vibrio cholerae 0139 in faecal specimens by coagglutination.

We compared the conventional culture method with the coagglutination (CoA) test for detecting V. cholerae 0139 antigen in a 4 h faecal enrichment culture. The CoA test reacted positively in all 13 culture positive stool specimens from patients with clinical cholera and negatively in all 23 culture negative specimens from non-diarrhoeal healthy controls. The test also did not show cross reaction with V. cholerae 01 antigen or with any of the enterobacterial antigens of the coliforms. The CoA test was found to be technically simple, rapid and reliable in diagnosing V. cholerae 0139 infection.

Monoclonal antibodies against Ogawa specific & Ogawa-Inaba common antigenic determinants of Vibrio cholerae O1 & their diagnostic utility.

Monoclonal antibodies to Ogawa-Inaba common antigenic determinant and Ogawa specific antigenic determinant of V. cholerae belonging to the serogroup O1 were generated from BALB/c mice immunized with V. cholerae O1 Eltor Ogawa strain. Reactivity and specificities of the monoclonal antibodies were examined by slide agglutination method. The monoclonal antibodies agglutinated all the V. cholerae O1 strains tested but did not agglutinate with any of the other currently recognized 140 serogroups of V. cholerae non-O1 as well as with a variety of other enteric pathogens. Diagnostic utility of the MAbs produced in this study as compared to polyclonal O1 and monospecific antisera showed that the MAbs were as good as the latter for serological diagnosis of the two serotypes of V. cholerae O1.

Evaluation of potency of inactivated cholera vaccine by mouse protection assay & antibody induction method.

Twenty one batches of whole cell inactivated cholera vaccine manufactured at Central Research Institute, Kasauli were evaluated for potency by mouse protection assay (MPA) and antibody induction method. In the antibody induction method the sera of immunized mice were screened for the presence of antibodies against Vibrio cholerae by microagglutination (MA) test and IgG ELISA. The number of organisms estimated by MPA were correlated with agglutinating and neutralizing antibodies against individual serotypes by MA and ELISA respectively. Correlation coefficient(r) of 0.692 and 0.815 were observed for the titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Ogawa. Similarly r values of 0.925 and 0.849 were observed for titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Inaba. Antibody induction method can be as an alternative method for determining the potency of inactivated cholera vaccine.

A monoclonal antibody-based dot-blot ELISA diagnostic kit for the detection of Vibrio cholerae 01 in stools of diarrheic patients and household contacts.

A "cholera diagnostic kit" was developed for sensitive, specific, rapid, and inexpensive detection of Vibrio cholerae 01. The monoclonal antibody specific to antigen A of Vibrio cholerae 01 was used as an antigen detection reagent and the principle of dot-blot ELISA was adopted. The kits were used in seven Regional Medical Sciences Centres, Ministry of Public Health, located at various regions of Thailand where diarrhea occurs frequently. Diagnostic efficiency of the kits in the detection of Vibrio cholerae 01 from rectal swabs of the diarrheic patients and their household contacts was evaluated in comparison with the conventional culture method. The two methods were found to have excellent degree of agreement (kappa values > 95%). The dot-blot ELISA has several advantages over the culture methods, ie rapid (dot-blot ELISA takes 1-2 hours while the culture method takes at least two days) and inexpensive. It requires no sophisticated equipment. The procedure is not complicated thus it is easy to train personnel. The diagnostic kits are recommended for use in the detection of severe diarrhea caused by V. cholerae 01 not only in hospitals and health centres where adequate treatment of the patients is required as a life-saving measure but also for early recognition of cholera cases and their contacts so that other action, ie prevention and control of outbreaks and surveillance can be promptly implemented.

Caracterización de una cepa de vibrio cholerae 01 multirresistente, aislada de un caso de cólera en Chile / Characterization of a multiresistant vibrio cholerae 01 strain, isolated from a cholera case in Chile

This report characterizes a multiresistant Vibrio Cholerae 01 strain, isolated from a patient with cholera and investigates the mechanism of resistance. The analyzed strain was resistant to tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. The resistance was mediated by a 101 megadalton plasmid that was transferred to the resultant of a conjugation assay between the multiresistant V. Cholerae strain and E. coli C-600 used as receptor strain, that acquired the triple resistance of the parental strain. The resistant V. Cholerae strain had a Ogawa serotype, El Tor biotype and toxigenic capacity, demonstrated by ELISA and latex agglutination techniques. The biochemical features of the strain were identical to those of susceptible strains, except for the resistance to 10 and 150 ug o 129 vibriostatic factor. The emergence of plasmid mediated resistance to drugs of choice in the treatment of cholera must alert Chilean and Latin American health authorities, considering the cholera will continue affecting the region

La inmunología del cólera y la biología molecular de la toxina colérica: progresos recientes y perspectivas futuras / The immunology of the cholera and th molecular biology of toxins

Recientemente Vibrio cholerae ha llamado mucho la atención de los investigadores por ser un inmunógeno muy potente y, al mismo tiempo, un coadyuvante inmunomodulador de la respuesta inmunitaria en la mucosa intestinal, tanto para los antígenos que se administran mezclados como los ligados covalentemente a la toxina colérica. La inmunopatogenia del cólera es un fenómeno complejo. En este artículo se comunican los resultados preliminares de experimentos realizados con ratas de laboratorio para conocer la respuesta intestinal de la IgA en los roedores y los humanos

Pasteur oral cholera vaccine: studies of reactogenicity, clinical acceptability and immunogenicity in human volunteers.

Pasteur cholera vaccine consists of isolated antigenic fractions from V. cholerae El Tor Ogawa and Inaba. Enteric coated microgranules were prepared from antigen lyophilisate. Three doses of this vaccine were administered orally to 19 healthy young Thai adults at one week intervals. None of the volunteers experienced untowards reactions. The vibriocidal antibody responses manifested a significant antibody rise (> or = 4 fold) to serovar Inaba in 8 vaccinees (42.1%) and Ogawa in 4 (21.1%). Five and 6 vaccinees (26.3% and 31.6%) showed a > or = 4 fold rise of IgG and IgA anti-LPS, respectively.